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Fluorescence imaging has become a powerful tool for measuring protein mobility and is particularly suited to probing the dynamics of protein-protein interactions in inclusion bodies. Two independent labs have just exploited these techniques to probe the aggregates formed by polyglutamine-expanded (polyQ) proteins such as those found in the neurodegenerative disorders spinocerebellar ataxia and Huntington’s disease. Their work appears in today’s Nature Cell Biology online.

Researchers at Michael Mancini’s lab at Baylor College of Medicine, Houston, Texas, studied inclusion bodies formed by the polyQ protein ataxin1. By measuring fluorescence recovery after photobleaching (see related news item) of green fluorescent protein (GFP)-ataxin1 chimeras in transfected cells, the authors determined how quickly free cytoplasmic protein could exchange with protein in polyQ aggregates. They found that aggregates comprise both fast- and slow-exchanging components with the speed of the exchange depending on the length of the polyQ expansion. Those proteins with 84 glutamines, for example, exchanged much more slowly than those with only two glutamines, such that 87 percent of inclusions with 84Q chimeras but only 19 percent of 2Q chimeras were classified as slow-exchanging. The authors found that the expansion length was also a predictor of the size of inclusions. 2Q, 30Q, and 84Q expansions result in large inclusions in 4.4, 12.3, and 18.2 percent of infected cells, respectively.

Stenoien, et al., also examined mobility in inclusions containing ubiquitin, CREB-binding protein (CBP), and the LMP2 subunit of the proteasome core, proteins that have been shown previously to associate with polyQ inclusions. Low and high amounts of ubiquitin correlated with fast- and slow-exchange of ataxin-84Q, respectively, whereas the effect of LMP2 on ataxin mobility was just the opposite. These results suggest that the ubiquitin-proteasome degradation pathway has a significant influence on the dynamics of these aggregates. Interestingly, LMP2 and CBP mobility was faster than ataxin 1, even in aggregates where the latter exchanged very slowly with its cytoplasmic form, indicating that at least some proteins are not irreversibly trapped in these inclusions.

This was also the conclusion reached by Soojin Kim and colleagues working under the direction of Richard Morimoto at Northwestern University, Evanston, Illinois. Using similar techniques they studied the interaction between polyQ proteins, the TATA binding protein (containing a 37-glutamine stretch) and the chaperone Hsp70, which has been shown previously to attenuate the toxicity associated with polyQ expansions. They found that only 17 percent of the TATA binding protein found in aggregates was readily diffusible, while in contrast over 70 percent of Hsp70 associated with aggregates was mobile. In fact, the rate of diffusion of Hsp70 was comparable to that observed between the chaperone and unfolded protein substrates in the nucleolus. This fact, coupled with the observation that Hsp70 lacking its substrate binding domain diffused even faster, suggests some active role for the chaperone, perhaps in inhibiting aggregate growth.

One caveat to these and similar studies, is that not all aggregates and inclusion bodies may behave similarly. Previous work by Chai et al, for example, has shown that aggregates of ataxin 1 and ataxin 3 behave quite differently, the latter tightly sequestering CBP.

“These are elegant papers,” says Henry Paulson, University of Iowa. “They extend the idea that these inclusions are complex. They do have aggregated protein, that seems very clear, but they also have proteins that associate in a dynamic way.” But there are differences. “We found that ataxin3 behaved like it was stuck whereas ataxin 1 had a lot of dynamic behavior,” recalls Paulson, “and Mancini’s group really shows that ataxin1 protein is a dynamic protein. It has become very clear of the last couple of years that researchers must investigate these polyglutamine domains within the context of the protein. Some of the differences that exist in disease clinically, may relate to differences in the actual proteins in which polyglutamine occurs.”—Tom Fagan

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Two new papers demonstrate several interesting findings that demonstrate that components of aggregated, polyglutamine-containing inclusions seen in certain neurodegenerative diseases are highly dynamic. Stenoien, et al., used fluorescence recovery after photobleaching (FRAP) to show that the ataxin 1 protein, when tagged with green fluorescent protein (GFP) can be present in small or large aggregates within cells. With either short or long polyglutamine stretches, the protein aggregates though there were generally smaller inclusions with transfection of the shorter polyglutamine stretches and larger inclusions with the longer stretches. Interestingly, the ataxin 1 present in aggregates was shown to be highly dynamic with a half-life in small aggregates of only seconds and a half life in larger aggregates of 20 seconds. This dynamic equilibrium of misfolded proteins (felt previously to be somewhat static) is fascinating and suggests that molecular chaperones for these proteins play an important role in both the aggregation and disaggregation process. Further supporting that idea, the authors also found that the presence of ubiquitin associated with aggregates was linked with a longer half life of aggregates and the presence of a proteasome component was linked was a shorter half life.

Kim, et al., used similar approaches such as FRAP as well as fluorescence loss in photobleaching (FLIP) and found that the interaction between molecular chaperones for polyglutamine-containing proteins such as Hsp70 were very different than the interaction between the polyglutamine-containing aggregates with themselves. They showed that while the polyglutamine aggegrates were “relatively” stable, the interactions of Hsp70 were very transient with the aggregates. They also found different aggregate dynamics and interaction with other proteins associated with the aggregates. These results demonstrate highly dynamic interactions with molecular chaperones and aggregates and that different classes of proteins interact in distinct ways with aggregates.

In total, these two papers show that modification of aggegrated proteins in neurodegenerative diseases (e.g. Aβ, tau, polyglutamine proteins, synuclein, superoxide dismutase) is likely to be a dynamic process and that proteins are not irreversibly sequestered into these structures. Further, the results suggest that molecular chaperones (e.g. apoE, ubiquitin, heat shock proteins, etc.) and their modifiers would be good drug targets in that modification of their expression or function may be able to reverse the process of protein aggregation and its consequences faster than we previously thought.